Nuclear Subfractions Research Articles

Effect of 3-methylcholanthrene on nuclear subfractions in rat liver

Effect of 3-methylcholanthrene on nuclear subfractions in rat liver

Early postnatal development of calmodulin-dependent protein kinase II in rat brain

The postnatal levels of the α- (M r 50,000) and β- (M r 58,000/60,000) subunits of type II calmodulin-dependent protein kinase were investigated in cytosolic, cytoskeletal and nuclear subfractions of rat brain. Substantial amounts of the β-subunit were present neonatally, especially in the cytoplasm; both subunits increased markedly during the subsequent three weeks with α-preponderance in the cortex and midbrain. The development of the α-subunit was attenuated in the cerebellum and brain stem, resulting in a relative excess of the β-subunit. Primary neuronal cultures from fetal brain initially displayed low enzyme content, followed by an increase in the levels of the β- but not α-subunit. Regulation of the cellular content of type II calmodulin-dependent protein kinase may be relevant to neuronal differentiation.

Association of phosphorylated Simian virus 40 T-antigen with subnuclear fractions of infected and transformed cells

To define the roles of subnuclear structure in SV40 infection, the relative distribution of T-antigen (T-ag) in various subnuclear fractions obtained from both lytically infected and transformed African green monkey kidney cells was determined. Depending on the differential sensitivity of nuclear T-ag to extraction by salt and detergent, nuclear T-ag could be separated into nucleoplasmic T-ag, salt-sensitive T-ag and matrix-bound T-ag subclasses. At least fivefold less matrix-bound T-ag was found in transformed cells than in lytically infected cells. While a cAMP-independent protein kinase was detected in the nuclear matrix, the matrix-bound T-ag (94K) could not be phosphorylated in vitro. The removal of cellular chromosomes by DNase caused changes in the interaction of T-ag with nuclear components. The results suggest that the compartmentalization of nuclear T-ag may be determined by its interaction with host chromosomes.

Exclusive nuclear location of estrogen receptors in Squalus testis.

An estrogen (E)-binding molecule having both occupied and unoccupied sites is restricted to nuclear subfractions in the testis of the spiny dogfish (Squalus acanthias). We investigated the hypothesis that a species characterized by high body-fluid osmolarity (1010 mosM) has an estrogen receptor (ER) that binds to chromatin with high affinity and consequently resists redistribution during tissue processing. Although the steroid binding and sedimentation properties of the Squalus nuclear ER conformed to those of classical ER, its elution maximum from DNA-cellulose was unusually high (0.55 M NaCl). A tendency to adhere tightly to cell nuclei was reflected in the high salt concentration (0.43 M KCl) required to extract 50% of the receptors from the nuclear compartment during homogenization and in the stability of the nuclear ER population in the presence of high concentrations of a nonionic solute (urea) or increased buffer volume. Mixing and redistribution experiments showed that nuclear ER could be quantitatively and qualitatively measured in cytosolic extracts, ruling out the possibility that soluble receptors were being masked. Although Squalus oviduct ER was similar to that of testis, ER in the testis and liver of a related elasmobranch (Potamotrygon) that maintains osmotic equilibrium at 300 mosM more closely resembled mammalian ER in its elution maximum from DNA-cellulose (0.22 M NaCl) and cytosolic/nuclear ratios in low-salt buffers. We conclude that Squalus testis has a single ER pool located exclusively in the nuclear compartment. These observations support a revised concept of steroid action and further indicate that the chromatin affinity of the hormone-ER complex is an important factor in determining subfractional distribution during tissue processing.

Insulin and the regulation of isolated nuclei and nuclear subfractions: potential relationship to mRNA metabolism.

Diabetes/Metabolism ReviewsVolume 1, Issue 1-2 p. 119-137 Article Insulin and the regulation of isolated nuclei and nuclear subfractions: Potential relationship to mrna metabolism I. D. Goldfine, I. D. Goldfine Cell Biology Research Laboratory, Harold Brunn Institute, Mount Zion Hospital and Medical Center, San Francisco, CA 94120 and Departments of Medicine, Physiology, and Pathology, University of California, San Francisco, CA 94120Search for more papers by this authorF. Purrello, F. Purrello Cell Biology Research Laboratory, Harold Brunn Institute, Mount Zion Hospital and Medical Center, San Francisco, CA 94120 and Departments of Medicine, Physiology, and Pathology, University of California, San Francisco, CA 94120Search for more papers by this authorR. Vigneri, R. Vigneri Cell Biology Research Laboratory, Harold Brunn Institute, Mount Zion Hospital and Medical Center, San Francisco, CA 94120 and Departments of Medicine, Physiology, and Pathology, University of California, San Francisco, CA 94120Search for more papers by this authorG. A. Clawson, G. A. Clawson Cell Biology Research Laboratory, Harold Brunn Institute, Mount Zion Hospital and Medical Center, San Francisco, CA 94120 and Departments of Medicine, Physiology, and Pathology, University of California, San Francisco, CA 94120Search for more papers by this author I. D. Goldfine, I. D. Goldfine Cell Biology Research Laboratory, Harold Brunn Institute, Mount Zion Hospital and Medical Center, San Francisco, CA 94120 and Departments of Medicine, Physiology, and Pathology, University of California, San Francisco, CA 94120Search for more papers by this authorF. Purrello, F. Purrello Cell Biology Research Laboratory, Harold Brunn Institute, Mount Zion Hospital and Medical Center, San Francisco, CA 94120 and Departments of Medicine, Physiology, and Pathology, University of California, San Francisco, CA 94120Search for more papers by this authorR. Vigneri, R. Vigneri Cell Biology Research Laboratory, Harold Brunn Institute, Mount Zion Hospital and Medical Center, San Francisco, CA 94120 and Departments of Medicine, Physiology, and Pathology, University of California, San Francisco, CA 94120Search for more papers by this authorG. A. Clawson, G. A. Clawson Cell Biology Research Laboratory, Harold Brunn Institute, Mount Zion Hospital and Medical Center, San Francisco, CA 94120 and Departments of Medicine, Physiology, and Pathology, University of California, San Francisco, CA 94120Search for more papers by this author First published: 1985 https://doi.org/10.1002/dmr.5610010107Citations: 12AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume1, Issue1-21985Pages 119-137 RelatedInformation

Thyroxine preferentially stimulates transcription by isolated neuronal nuclei in the developing rat brain cortex

The administration of thyroxine to neonatal rats stimulates RNA synthesis by neuronal nuclei isolated from the developing rat brain cortex. Glial nuclei are relatively resistant to thyroxine treatment. The activity of neuronal RNA polymerase II is particularly stimulated by the hormone. Thyroxine also affects neuronal chromatin structure as shown by changes in the relative proportion of different subnuclear fractions obtained by gentle micrococcal nuclease digestion of nuclei from hormone-treated rats.

Distribution of thiopyronine in subnuclear fractions of human fibroblast cells: extend of binding to nDNA in vivo

Distribution of thiopyronine in subnuclear fractions of human fibroblast cells: extend of binding to nDNA in vivo

Retinoid binding to nuclei and microsomes of rat testes interstitial cells: I — Mediation of retinoid binding by cellular retinoid-binding proteins

Purified cellular retinol-binding protein (cRBP) and cellular retinoic acid-binding protein (cRABP) mediated the transfer of their respective 3H-ligands to nuclei and microsomes of retinol-sufficient rat testes interstitial cells in a saturable and reversible manner. Crossover experiments utilizing [ 3H]retinol·cRBP/retinoic acid·cRABP and [ 3H]retinoic acid·cRABP/retinol·cRBP did not result in dissociation of the labeled ligand from either the nuclear or microsomal fraction. Further, binding of free [ 3H]retinol or [ 3H]retinoic acid was not saturable nor was binding detectably reduced in the presence of unlabeled retinoids. When nuclei and microsomes were prepared from retinol-deficient tissues, cRBP-mediated binding of [ 3H]retinol was also saturable and reversible. However, total binding was significantly increased in both cell fractions. Similarly, cRABP-mediated binding of [ 3H]retinoic acid was significantly higher in retinol-deficient nuclei and microsomes. Extraction of cRBP- or cRABP-mediated nuclear-bound 3H-retinoids with NaCl indicated that 50% of the retinol and retinoic acid were removed with 0.14 M NaCl. Retinol binding in nuclear subfractions including the 0.14 M NaCl fraction was pronasesensitive, RNase and DNase insensitive; retinoic acid was pronase- and RNase-sensitive, and DNase-insensitive. Nuclear binding of free retinoids was <15% sensitive to NaCl extraction with the major portion being retained in the nuclear membrane. These data indicate that nuclear and microsomal binding of retinoic acid as well as retinol is mediated by its transport protein. Further, microsomes as well as nuclei are capable of specific binding of retinol and retinoic acid when binding is mediated by cRBP and cRABP, respectively. Retinoid binding in nuclei appears to form a complex which is predominantly protein, suggesting that retinoids do not, upon initial binding, interact directly with DNA.

The effect of 5-fluoroorotic acid on the early labelling of nucleotides and RNA in whole liver and of RNA in subnuclear fractions in rats given 3H-orotic acid.

Male white rats were given either orotic acid or 5-fluoroorotic acid 60 min, and 3H-orotic acid 3 1/2 min before sacrifice. Liver nucleotides were analyzed by isotachophoresis. The (F)UTP pool increased to the same extent in both groups and showed the same specific labelling. The RNA synthesis, as measured by nmol of UTP entering RNA-UMP/g wet liver tissue was significantly lower in fluoroorotic acid treated rats. In the nucleolar fraction there was an increase in the RNA/DNA ratio, a decrease in the specific RNA labelling and an essentially unaltered specific labelling of RNA/microgram/DNA. The effect on the specific labelling of RNA/microgram DNA was the same in the nucleolar fraction and the nucleolar-free pellet, suggesting that the transcription of RNA in these two fractions proceeded in a similar way. There was a slight increase in the specific RNA labelling of the supernatant subnuclear fraction, containing low-molecular weight RNA. Incorporation into UDP-hexoses and UDP-N-acetylhexosamine was unchanged.

Glucocorticoid regulation of transcription at an amplified, episomal promoter.

The mouse mammary tumor virus long terminal repeat (MMTV LTR) has been introduced into cultured murine cells, using the 69% transforming fragment of bovine papilloma virus type 1 (BPV). Transformed cells contain up to 200 copies of the chimeric molecules per diploid genome. The restriction endonuclease map of the acquired recombinants, as well as the physical structure of the DNA, indicates that the LTR-BPV molecules present in these cells occur exclusively as unintegrated, extrachromosomal episome. When a 72-base pair direct repeat "enhancer" element (derived from the Harvey sarcoma retrovirus) was included in the MMTV LTR-BPV chimeric plasmids, DNA acquired through transfection, with a single exception, was integrated or rearranged or both. The transcriptional potential of the episomal MMTV promoter present in these cells was tested in two ways. First, steady-state levels of MMTV-initiated RNA were measured by quantitative S1 mapping. Second, the relative number of transcription complexes initiated in vivo was determined by using a subnuclear fraction highly enriched for MMTV-BPV minichromosomes in an in vitro transcription extension assay. Both approaches showed that the MMTV LTR present in the episomal state was capable of supporting glucocorticoid hormone-regulated transcription. We have therefore demonstrated the hormone response for the first time in a totally defined primary sequence environment. Significant differences both in the basal level of MMTV-initiated transcription and in the extent of glucocorticoid induction were observed in individual cell lines with similar episomal copy numbers. These phenotypic variations suggest that epigenetic structure is an important component of the mechanism of regulation.

Intracellular localization of adenovirus type 5 tumor antigens in productively infected cells

The intracellular localization of tumor antigens of human adenovirus type 5 (Ad5) during lytic infection of KB cells has been studied. The cells were pulse labeled with [ 35S]methionine early after infection and early proteins of 58,000 D (58K), 44K,19K,18.5K, and 14K detectable by immunoprecipitation with hamster antitumor serum were assayed for association with cytoplasm, nucleoplasm, chromatin, cytosol, cytoskeleton, and membranes. The 44,000 D (44K) tumor antigen encoded in early region IA (E1A: 0–4.4%) was recovered in approximately equal amounts from cytoplasmic and nucleoplasmic fractions of pulse-labeled cells and within the cytoplasmic compartment was found in the cytosol as well as associated with the cytoskeleton. The E1B-58K (E1B: 4.5–11.2%) antigen was also found to be associated with the cytoplasmic and nucleoplasmic fractions in approximately equal amounts but unlike the ElA-44K showed no affinity for cytoskeletons. Pulse-chase and immunofluorescence experiments suggested the 58K antigen accumulated in the nucleus late in infection. The E1B-19K antigen was found almost exclusively associated with the membrane fraction of infected KB cells and was resolved in polyacrylamide gels into two related species of 18.5K and 19K. Immunofluorescence studies on the ElB 18.5–19K doublet suggested that within a population of infected HeLa cells a small minority seemed to be expressing copious amounts of stainable antigen. Cell fractionation and immunofluorescence studies showed that the E4-14K antigen was a nuclear protein and the only antigen in this study which showed a significant association with a nuclear subfraction composed almost entirely of histones. The implications of these findings for the roles of the Ad5 tumor antigens in lytic infection and transformation are discussed.

Dynamic domains of DNA polymerase alpha in regenerating rat liver.

Intranuclear redistributions of DNA polymerase alpha activity (the presumptive replicative enzyme) are observed preceding and during in vivo replication in regenerating rat liver. The most marked induction of in vitro enzyme activity occurs coincident with the onset of in vivo replication and is seen in the high salt resistant domain of the nuclear matrix. Moreover, DNA polymerase alpha endogenous and exogenous template activities begin to redistribute prereplicatively from bulk chromatin directly to the high salt resistant matrix. These prereplicative subnuclear events are detectable prior to the induction of total cellular DNA polymerase alpha and appear to continue throughout replication. Maximal percent recovery of the total nuclear activity on the high salt resistant matrix (approximately 30%) occurs coincident with and during the time when in vivo replication rates are maximum. At the conclusion of replication, DNA polymerase alpha activities shift back into the bulk chromatin and are no longer high salt resistant. In contrast, DNA polymerase beta (presumptive repair enzyme) endogenous and exogenous template activities are randomly distributed in the nuclear subfractions through the proliferative response. We conclude that DNA polymerase alpha is dynamically assembled or activated on the nuclear matrix and that this process is initiated before the onset of in vivo replication.

Effect of heparin on the subcellular fractionation of a polyamines rich organ (rat ventral prostate)

Rat ventral prostate is very rich in polyamines that, during homogenization, cause aggregation of subcellular organelles jeopardizing subfractionation studies of this organ. 1. 1) Heparin added in vitro to rat ventral prostate subcellular fractions to sequester polyamines exerts the following effects: a) minimizes aggregation of most membranous material and lowers its sedimentation rate; b) removes some ribosomes from the microsomal vesicles; c) causes disruption of nuclei and an apparent subnuclear fractionation. 2. 2) After removal of nucleic which interfere in fractionation, heparin was added to subfractionate the other cellular components. Heparin concentration and centrifugation conditions were tested to set up a reliable method of subcellular fractionation. 3. 3) The distribution of the marker enzymes galactosyltransferase (Golgi apparatus) and acid phosphatase (lysosomes and secretory granules) were checked to assess the usefulness of the method.

Base-pairing interactions between small nuclear RNAs and nuclear RNA precursors as revealed by psoralen cross-linking in vivo

The psoralen derivative 4′-aminomethyl-4,5′,8-trimethylpsoralen (AMT) reacts with base-paired regions of RNA and forms interstrand covalent cross-links. Since psoralens permeate living cells, they can be used to probe RNA-RNA interactions in vivo. We used AMT to investigate whether small nuclear RNAs are base-paired with high molecular weight nuclear RNA in the cell. Intact HeLa cells were treated with AMT, and high molecular weight RNA was isolated under denaturing conditions from nuclei or from subnuclear fractions. The presence of base-paired snRNAs in the high molecular weight nuclear RNA was examined by electrophoresis after photochemical reversal of the cross-links. We found snRNA U3 and 5.8S rRNA to be cross-linked to nucleolar RNA. In contrast, snRNA U1 was cross-linked to high molecular weight RNA in ribonucleo-protein particles containing hnRNA. The U1 base-paired to hnRNA was identified by its hybridization with a cloned U1 DNA sequence after reversal of the cross-links. These results demonstrate that U1 is base-paired with hnRNA in vivo, suggesting a role in mRNA processing.

Increase in nuclear cyclic GMP-dependent protein kinase following partial hepatectomy

The level of cyclic GMP-dependent protein kinase in the nucleus of rat liver was shown to increase 80% at 3 hr following partial hepatectomy while cyclic AMP-binding activity decreased 28%. Subnuclear fractionation demonstrated that the increase in cyclic GMP-dependent protein kinase was localized to the nucleolus and nucleoplasm, with no change in the extranucleolar particulate material. Cyclic AMP-binding activity was decreased in all subnuclear fractions under these conditions. At 16 hr following partial hepatectomy, the level of cyclic GMP-dependent protein kinase was not changed in the nucleolus but was significantly increased in the nucleoplasm, while cyclic AMP-binding activity was slightly increased in the nucleolus and decreased in the nucleoplasm.

A possible role of rabbit heart cytosol tocopherol binding in the transfer of tocopherol into nuclei

An alpha-tocopherol-binding macromolecule was isolated from the heart cytosol of rabbits fed for 1 month with an alpha-tocopherol-deficient diet. The amount of [3H]-tocopherol bound to nuclear chromatin was increased when the alpha-tocopherol-deficient heart nuclei were incubated in the presence of [3H]tocopherol-cytosol complex. In this condition, large amounts of [3H]tocopherol were associated with a subnuclear fraction that contained non-histone acidic proteins.

The specific binding of estrogens and androgens to the nuclear matrix of sex hormone responsive tissues.

Specific sex steroid hormone binding sites have been identified in a discrete nuclear subfraction that is chromatin-depleted and salt-insoluble. This structure, referred to as the nuclear matrix, contains a residual pore complex-lamina, remnants of an internal network, and a residual nucleolus. The nuclear matrix of an estrogen-responsive tissue (chicken liver) and of an androgen target tissue (rat ventral prostate) contains binding sites for estradiol and dihydrotestosterone, respectively. The binding of steroids to these sites is saturable, high affinity (Kd approximately 10(-9) M), steroid-specific (liver binds estrogens, prostate binds androgens), and heat- and pronase-sensitive. The levels of these matrix-associated steroid binding sites change in response to manipulation of the hormonal status of the animal. Thus, the liver nuclear matrix of laying hens contains a significant number of estradiol binding sites, whereas that of untreated chicks or roosters contains very few sites. Treatment of chicks or roosters with a dose of estrogen that stimulates the liver to synthesize vitellogenin leads to a marked increase in the number of estradiol binding sites associated with the liver nuclear matrix. In the rat ventral prostate, the nuclear matrix-associated binding sites for dihydrotestosterone that are present in intact adults essentially disappear within 24 h after castration. Androgen replacement therapy restores, within 1 h, the number of matrix binding sites for dihydrotestosterone to normal levels. Inhibitors of proteolysis (phenylmethylsulfonyl fluoride) are essential for the protection of these androgen binding sites. Conditions that lead to the solubilization of the internal network material of the nuclear matrix also result in the extraction of 70 to 85% of the specific dihydrotestosterone binding sites from the matrix; a limited number of binding sites still remain associated with the peripheral lamina.

Antigenic properties of the loosely bound subnuclear fraction of rat brain

Antibodies against the loosely bound subnuclear fraction (0.35 M NaCl-extractable subnuclear fraction) of rat brain were raised in rabbits, and the distribution of the main antigenic determinants was followed among subcellular fractions of nervous tissue and among homologous nuclear preparations from different tissues. By immunofluorescence a localization restricted to the nucleus was observed, and by microcomplement fixation the antigens appeared to specifically enrich the fraction under examination, being poorly detectable in cytosol, nuclear sap, or deoxyribonucleoproteins of rat brain. No significant cross-reaction was observed by complement fixation with homologous preparations from muscle, liver, kidney, spleen, lung, or thymus of rat, whereas the 0.35 M NaCl-extracted subnuclear fraction from rat testis exhibited an immunoreactivity, although lower than that for brain proteins. After trypsin or ribonuclease treatment, the main antigenic determinants appeared to be protein in nature. The subnuclear fraction under examination, which is believed to be relevant to gene regulation, appears to contain protein antigens mainly concentrated in the nervous system.

Localization of the loosely bound nuclear proteins of rat brain: an immunocytochemical ultrastructural study.

Antibodies against the loosely bound subnuclear protein fraction (0.35 M NaCl-extractable subnuclear fraction) of rat brain were raised in rabbits, and the ultrastructural distribution of the antigenic determinants in rat cerebellum was studied using the unlabeled antibody peroxidase-antiperoxidase (PAP) method. A localization restricted to the nucleus was observed. Both neuronal and neuroglial nuclei exhibited antigens, whereas nuclei of pericytes and endothelial cells did not. The immunoreaction product was homogeneously distributed in dispersed chromatin and was absent from condensed chromatin, suggesting that the antigens were confined to the active regions of the genoma. The outer nuclear membrane and the nucleolus appeared to be free of the antigens, while a perinucleolar ring of immunoreaction was detectable. Liver preparations showed a nuclear reaction markedly weaker than the one for brain nuclei. Adsorption of the serum with isolated liver nuclei nullified the reactivity in liver tissue, whereas a sharp reaction was still observed in the cerebellum, indicating the subcellular reaction under examination to contain antigens specifically concentrated in the nervous system or unique to the brain.

Subnuclear fractionation by mild micrococcal-nuclease treatment of nuclei of different transcriptional activities causes a partition of expressed and non-expressed genes.

Extremely mild treatment with micrococcal nuclease of isolated nuclei yields subnuclear fractions in which the majority of RNA polymerase II transcriptional complexes formed in vivo are segregated [Tata & Baker (1978) J. Mol. Biol. 118, 249-272]. We now describe different approaches followed to established whether or not the nuclei are thus resolved into transcribed and non-transcribed DNA. First, we have compared the sensitivity to deoxyribonuclease I, which is known to digest preferably expressed genes as present in nuclei or chromatin, of three micrococcal-nuclease-derived fractions from nuclei of different transcriptional activities. In transcriptionally active nuclei (rat liver, hen liver and oviduct, and Xenopus liver), the DNA in a polynucleosomal fraction comprising 6-15% of DNA and the majority of template-engaged RNA polymerase II (fraction P2) was 10-50 times as sensitive to deoxyribonuclease I as the DNA in the other two fractions (fractions P1 and S, comprising 78-88% of total nuclear DNA as large polynucleosomal aggregates and 2-6% of DNA mostly as mononucleosomes, respectively). In transcriptionally inactive nuclei obtained from hen erythrocytes, micrococcal nuclease did not separate DNA into fractions exhibiting such differential sensitivities. Second, we have monitored the partition of an expressed gene. Hybridization of complementary DNA to Xenopus albumin mRNA revealed a 5-10-fold enrichment of the albumin (but not the globin) gene in the P2 fraction of nuclei from Xenopus liver in which this gene is fully expressed. Third, a large part of the nascent rapidly labelled RNA synthesized in vivo in rat liver nuclei was recovered in the micrococcal-nuclease-derived fraction that is more susceptible to digestion with deoxyribonuclease I. It is concluded that mild micrococcal-nuclease treatment of nuclei causes their separation into transcribed and non-transcribed DNA as determined by a number of very different criteria.